Stereochemical assignment of intermediates in the rifamycin biosynthetic pathway by precursor-directed biosynthesis

Natural and semisynthetic rifamycins are clinically important inhibitors of bacterial DNA-dependent RNA polymerase. Although the polyketide-nonribosomal peptide origin of the naphthalene core of rifamycin B is well established, the absolute and relative configuration of both stereocenters introduced by the first polyketide synthase module is obscured by aromatization of the naphthalene ring. To decode the stereochemistry of the rifamycin polyketide precursor, we synthesized all four diastereomers of the biosynthetic substrate for module 2 of the rifamycin synthetase in the form of their N-acetylcysteamine (SNAC) thioester. Only one diastereomer was turned over in vivo into rifamycin B, thus establishing the absolute and relative configuration of the native biosynthetic intermediates.     

Synthesis and stereochemical assignment of (+)-chamuvarinin

A stereocontrolled total synthesis of (+)-chamuvarinin, isolated from the root extract of Uvaria Chamae, utilizes a convergent modular strategy to construct the adjacently linked C15-C28 ether array, followed by a late-stage Julia-Kocienski olefination to append the butenolide motif. This constitutes the first total synthesis of (+)-chamuvarinin, defining the relative and absolute configuration of this unique annonaceous acetogenin.     

Identification of a tetraene-containing product of the indanomycin biosynthetic pathway

The indanomycin biosynthetic gene (idm) cluster was recently identified from Streptomyces antibioticus NRRL 8167. The disruption of one of these genes, idmH, and the increased production of a previously unreported metabolite in this mutant is reported. The structure of this compound was elucidated and was shown to possess a linear tetraene. This metabolite is not a logical biosynthetic intermediate of indanomycin but instead is likely an alternate product of the pathway.     

Synthesis and stereochemical assignment of brasilibactin A

[structure: see text] Brasilibactin A, a naturally occurring siderophore related to the mycobactins, has been synthesized in six steps. Use of asymmetric titanium-mediated aldol reactions allowed the preparation of both diastereomers from a common synthetic intermediate, thus allowing the relative stereochemistry of the natural product to be assigned. Brasilibactin A exhibits no inhibition of histone deacetylases (HDACs) in spite of the N-formyl-N-hydroxy lysine moiety that is expected to affect the activity of these metal-dependent lysine-modifying enzymes.     

Synthesis of (±)-cyclic dehypoxanthine futalosine, the biosynthetic intermediate in an alternative biosynthetic pathway for menaquinones

The first synthesis of (±)-cyclic dehypoxanthine futalosine (cyclic DHFL), a biosynthetic intermediate in the futalosine pathway for menaquinones operating in microorganisms, has been achieved. Efficient growth of the Streptomyces coelicolor mutant, which lacks the cyclic DHFL synthetase gene (mqnC gene) was observed in the presence of synthetic (±)-cyclic DHFL.     

Synthesis and stereochemical assignment of (+)-Cladospolide D

Cladospolides A-D are 12-membered, α,β-unsaturated lactones isolated from various species of Cladosporium. Cladospolide D is unique in its γ-keto functionality and possesses antifungal activity; however, the stereochemistry of Cladoapolide D was unknown. We report the asymmetric syntheses to generate both possible diastereomers of Cladospolide D. Two regioselective cross-metatheses were applied to form the carbon skeleton, and the two olefins were differentiated by Michael addition, hydrogenation, and elimination. Later, the macrocycle was achieved through the Yamaguchi protocol. After comparing the spectroscopic data of the synthetic Cladospolide D with the reported values, the stereochemistry of Cladospolide D is confirmed as (2E,5R,11S).     

5-enolpyruvylshikimate 3-phosphate synthase: chemical synthesis of the tetrahedral intermediate and assignment of the stereochemical course of the enzymatic reaction

A chemical synthesis of both diastereomers of the tetrahedral intermediate involved in 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) catalysis has been accomplished. Combination of methyl dibromopyruvate with a protected shikimic acid derivative, phosphorylation, and lactonization afforded the intermediates (S)-15 and (R)-15, whose configurations were assigned by NMR. After introduction of the 3-phosphate group and deprotection, photoinitiated radical debromination of the dibromo analogues (S)-5 and (R)-5 was accomplished with tributyltin hydride in mixed aqueous solvents in the presence of surfactant to give the pyruvate ketal phosphates (R)-TI and (S)-TI, respectively. These compounds are stable at high pH, but decompose at pH 7 with a half-life of ca. 10 min. (R)-TI proved to be inert to EPSPS, while (S)-TI was converted by the enzyme to a mixture of 5-enolpyruvylshikimate 3-phosphate, shikimate 3-phosphate, and phosphoenolpyruvate. The demonstration that the enzymatic intermediate possesses the S-configuration at the ketal center confirms the mechanism as an anti addition followed by a syn elimination. Furthermore, it appears that the syn stereochemistry of the second step requires the phosphate leaving group to serve as the base in catalyzing its own elimination.     

Total synthesis and absolute stereochemical assignment of kibdelone C

Kibdelones are hexacyclic tetrahydroxanthones and potent anticancer agents isolated from an Australian microbe. Herein, we describe the synthesis of a chiral, nonracemic iodocyclohexene carboxylate EF ring fragment of the kibdelones employing an intramolecular iodo halo-Michael aldol reaction and its merger with an ABCD ring fragment to afford the congener kibdelone C.     

Synthesis and stereochemical assignment of DNA spore photoproduct analogues

Investigation of the DNA repair process performed by the spore photoproduct (SP) lyase repair enzyme is strongly hampered by the lack of defined substrates needed for detailed enzymatic studies. The problem is particularly severe because the repair enzyme belongs to the class of strongly oxygen-sensitive radical (S)-adenosylmethionine (SAM) enzymes, which are notoriously difficult to handle. We report the synthesis of the spore photoproduct analogues 1 a and 1 b, which have open backbones and are diastereoisomers. In order to solve the problem of stereochemical assignment, two further derivatives 2 a and 2 b with closed backbones were prepared. The key step of the synthesis of 2 a/b is a metathesis-based macrocyclization that strongly increases the conformational rigidity of the synthetic spore photoproduct derivatives. NOESY experiments of the cyclic isomers furnished a clear cross-peak pattern that allowed the unequivocal assignment of the stereochemistry. The results were transferred to the data for isomers 1 a and 1 b, which were subsequently used for enzymatic-repair studies. These studies were performed with the novel spore photoproduct lyase repair enzyme from Geobacillus stearothermophilus. The studies showed an accordance with a recent investigation performed by us with the spore photoproduct lyase from Bacillus subtilis, in that only the S isomer 1 a is recognized and repaired. The ability to prepare a defined functioning substrate now paves the way for detailed enzymatic studies of the SP-lyase lesion recognition and repair process.     

Synthesis and stereochemical assignment of geraniol- and nerol-derived Cygerol enantiomers

The enantiomers (up to 99% ee) of both geraniol- and nerol-derived 2-cyclohexyl-5,9-dimethyldeca-4,8-dienoic acid, the active ingredient of the wound healing medication Cygerol, were prepared via a low-temperature alkylation, basic hydrolysis, derivatization with (S)-4-benzyloxazolidin-2-one and chromatographic separation steps. The absolute configuration of stereocenters in the antipodes having an (E)- or (Z)-geometry of the internal double bond was determined based on characteristic ¹H NMR signals of the corresponding (S)-4-benzyloxazolidin-2-one-derived imides and on conversion to the known diethyl (S)-2-cyclohexylsuccinate and (S)-2-cyclohexylbutane-1,4-diol with reported specific rotations.     

Spontaneity in the patellamide biosynthetic pathway

Post-translationally modified ribosomal peptides are unusual natural products and many have potent biological activity. The biosynthetic processes involved in their formation have been delineated for some, but the patellamides represent a unique group of these metabolites with a combination of a macrocycle, small heterocycles and d-stereocentres. The genes encoding for the patellamides show very low homology to known biosynthetic genes and there appear to be no explicit genes for the macrocyclisation and epimerisation steps. Using a combination of literature data and large-scale molecular dynamics calculations with explicit solvent, we propose that the macrocyclisation and epimerisation steps are spontaneous and interdependent and a feature of the structure of the linear peptide. Our study suggests the steps in the biosynthetic route are heterocyclisation, macrocyclisation, followed by epimerisation and finally dehydrogenation. This study is presented as testable hypothesis based on literature and theoretical data to be verified by future detailed experimental investigations.     

Total synthesis of dipiperamide A and revision of stereochemical assignment

The first total synthesis of dipiperamide A has been achieved by employing a solid-state photodimerization of an (E)-cinnamic acid derivative. This critical step results in the cyclobutane ring, which exists in the natural product, with full control of the regio- and stereochemistry at the four stereogenic centers. Results from these studies indicate that the proposed stereostructure of natural dipiperamide A should be revised to the structure originally assigned to dipiperamide B.     

Relative stereochemical assignment of C-33 and C-35 in the antibiotic gladiolin

Gladiolin is a macrolide antibiotic isolated from Burkholderia gladioli BCC0238 with promising activity against Mycobacterium tuberculosis, including several multidrug resistant strains. The configuration of all but one of the stereogenic centers of gladiolin has previously been elucidated using a combination of NOESY NMR experiments and predictive sequence analysis of the polyketide synthase responsible for its assembly. However, it was not possible to assign the configuration of the C-35 methyl group using such methods. Here we report the synthesis of C-33/C-35-syn and C-33/C-35-anti mimics of the C-30 to C-38 fragment of gladiolin from (R) and (S)-citronellol, respectively. Comparison of HSQC NMR data for the mimics and the natural product showed that the C-35 methyl is anti to the C-33 hydroxyl group, indicating that gladiolin has the 35S configuration.     

Synthesis and complete stereochemical assignment of psymberin/irciniastatin A

We describe a concise and flexible synthetic avenue for the preparation of compounds with structures relevant to those proposed for the novel marine-derived differential cytotoxins psymberin and irciniastatin A. Our efforts led to their complete stereochemical assignment and the notion that psymberin and irciniastatin A are identical compounds. Our total synthesis features an interesting termini-differentiating lactolization of a dialdehyde obtained from a C2-symmetrical bis-olefin precursor, a mild platinum-catalyzed hydrolysis of an epimerizable nitrile, a novel protocol to prepare sensitive methyl imidates, and a one-pot conversion of these imidates to N-acyl aminals. Starting from fragments 5-7 (prepared in 7-8 steps each, 30-49% overall yield) the synthesis of psymberin/irciniastatin A was completed in an additional 9 steps and 30% yield (17 steps longest linear sequence, 8.9% overall).     

Partial stereochemical assignment of quartromicins A3 and D3

[structure: see text]. A partial stereochemical assignment of quartromicins A3 (R = alpha-D-galactosyl) and D3 (R = H, M+ = Na+, K+, Ca+) is presented.     

Total synthesis and stereochemical assignment of the spiroisoxazoline natural product (+)-calafianin

[STRUCTURE: SEE TEXT] Synthesis of the spiroisoxazoline natural product (+)-calafianin is reported using asymmetric nucleophilic epoxidation and nitrile oxide cycloaddition as key steps. Synthesis and spectral analysis of all calafianin stereoisomers led to unambiguous assignment of relative and absolute stereochemistry.     

Azedaralide: total synthesis, relative and absolute stereochemical assignment

Azedaralide, a potentially advanced intermediate for the total synthesis of various tetranortriterpenes, was constructed utilising the Fernández-Mateos protocol and assigned both relative and absolute stereochemistries. Both asymmetric aldol and classical chiral resolution attempts failed to deliver pure enantiomers whereas preparative chiral chromatography resolved racemic azedaralide with ease.     

Enantiomeric resolution,stereochemical assignment and toxicity evaluation of TPA enantiomers

Tetrahydro‐α ‐(1‐methylethyl)‐2‐oxo‐1(2H)‐pyrimidineacetic acid (TPA) is a critical intermediate in the synthesis of HIV protease inhibitors. A simple and efficient method for the separation and determination of TPA enantiomers was developed. The TPA was separated into its enantiomers with an enantiomeric purity of 99% using an HPLC system equipped with a Chiralpak OD‐H column. Semi‐preparative HPLC enantioseparations were carried out for further enrichment of the enantiomers. The validity of this method was evaluated on the basis of its precision, accuracy, linearity and recovery. The method was observed to be suitable for the rapid separation and semi‐preparation of TPA isomers. The separated enantiomers were identified by optical rotation and high‐resolution electrospray ionization mass spectrometry. Furthermore, the stereochemical structures of the TPA enantiomers were definitively confirmed using a combination of experimental and calculated electronic circular dichroism spectra. The toxicity of the separated pure enantiomers against Oryzias melastigma was evaluated using the median lethal concentration (LC₅₀) values. The results indicated that (S )‐(−)‐TPA is ~2.5 times more toxic than its enantiomorphism.